This work was supported by the NINDS Intramural Research Program.

1. NSC culture
NOTE: Manipulation of a human NSC line will be described below but any cell line can be used for this protocol. All cell culture work is performed in a biological safety cabinet.
<ol>
	<li>Coat a 96-well plate with basement membrane/extracellular matrix (ECM).
	<ol>
		<li>Thaw aliquot of ECM (Table of Materials), which will facilitate attachment of NSC, on ice. Dilute ECM to the appropriate concentration (generally 1:100) in 10 mL base medium (Table of...

<ol>
	<li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Toxicity Testing in the 21st century: A Vision and a Strategy. , (2007).</li><li>Llorens, J., Li, A. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biology. 7 (10), 100 (2006).</li><li>Cerbini, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+activator-like+effector+nuclease+(TALEN)-mediated+CLYBL+targeting+enables+enhanced+transgene+expression+and+one-step+generation+of+dual+reporter+human+induced+pluriopotent+stem+cell+(iPSC)+and+neural+stem+cell+(NSC)+lines.">Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluriopotent stem cell (iPSC) and neural stem cell (NSC) lines.</a> PLoS One. 10 (1), 0116032 (2015).</li><li>Lundholt, B. 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The authors have nothing to disclose.

The primary goal of this article was to describe a strategy that could efficiently and inexpensively identify compounds affecting cell growth in a low- to moderate-throughput screening. Two orthogonal techniques were utilized to assess cell number to increase confidence in the conclusions and offer additional insights that would not be available if only a single assay was used. One of the assays used a fluorescent cell imager to directly count tdTomato-positive cells and the second was dependent on the well-characterized...

One of the most important characteristics that needs to be determined for a chemical compound that has therapeutic potential is its toxicity to animal cells. This characteristic will determine whether a drug is a good candidate for more extensive study. In most instances, compounds with minimal toxicity are sought but there are situations in which a compound with the capacity to kill specific cell types is of interest, e.g., anti-tumorigenic drugs. Although whole animals are the best model systems to determine systemic toxicity, the cost and labor involved is prohibitive when more than a few compounds need to be tested. As such mammalian cell culture is generally used as the most efficient alternative1,2. Small to medium throughput drug screens are an important modality through which toxicity can be assessed in cell culture. These screens can be used to interrogate annotated libraries targeting individual signaling pathways. The general format of such a screen is to initially test all the compounds in the library at a single dose (generally 10 &#181;M) in an exploratory primary toxicity screen, and then perform an in-depth secondary dose response screen to fully characterize the toxicity profile of hits from the primary screen. The methods to implement this strategy will be described here and provide a quick, efficient, and inexpensive way to identify and characterize toxic compounds.
Multiple methods have been developed to assess cytotoxicity of small compounds and nanomaterial in mammalian cells3,4. It should be noted that certain materials can interact with the assay providing misleading results, and such interactions should be tested when characterizing hits from toxicity screens4. Cytotoxicity assays include trypan blue exclusion5, lactate dehydrogenase (LDH) release assay6, Alamar blue assay7, calcien acetoxymethyl ester (AM)8, and the ATP assay9. All these assays measure various aspects of cell metabolism which can serve as a proxy for cell number. While all offer benefits, tetrazolium salt-based assays such as 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis(2-methoxy-4-nitro-5-sulfopheny)-2H-tetrazolium-5-carboxyanilide inner salt (XTT)-1, and 4-(3-[4-Iodophenyl]-2-[4-nitrophenyl]-2H-5-tetrazolio)-1,3-benzene disulfonate (WST-1)10,11 provide good accuracy and ease of use at low cost. MTT, which will be used in this demonstration, is reduced to an insoluble formazan by a mitochondrial reductase and the rate of this conversion correlates strongly with cell number. This assay has been routinely utilized at both a small scale and for screening libraries with up to 2,000 compounds12. Direct counting of cells by a labeled marker offers another method to assess the cellular number, and unlike the MTT assay it can provide additional information about the dynamics of cellular growth. Several publicly available algorithms are available to perform automated cell count analyses and there are also proprietary algorithms that are part of software packages for imaging readers13,14. In this method description, a human neural stem cell (NSC) line that has been genetically edited to constitutively express tdTomato15 will serve as a test line to compare cellular viability results between an MTT assay and an automated cell counting assay in a screen assessing toxicity of 57 test compounds. Although the primary goal of this strategy was to identify and characterize toxic compounds, it has the additional benefit of potentially identifying growth inhibitory and growth enhancing compounds and thus provides an effective method for identifying drugs that can modulate cellular growth.

<table border="0" cellpadding="0" cellspacing="0" style="width:1292px;" width="1292">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B-27 (50X)</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">17504001</td>
			<td style="width:720px;">Neural stem cell medium component.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BenchTop pipettor</td>
			<td style="width:237px;">Sorenson Bioscience</td>
			<td align="right" style="width:119px;">73990</td>
			<td>Provides ability to pipette compound library into a 96-well plate in one shot.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BioLite 96 well multidish</td>
			<td style="width:237px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">130188</td>
			<td>Any 96 well cell culture plate will work.&#160; We use these in our work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture microscope</td>
			<td style="width:237px;">Nikon</td>
			<td style="width:119px;">Eclipse TS100</td>
			<td>Visual inspection of cells to ensure proper density.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytation 5/ Imaging reader</td>
			<td style="width:237px;">BioTek</td>
			<td style="width:119px;">CYT3MFV</td>
			<td>Used for cell imaging and absorbance readings.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:237px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">610420010</td>
			<td>Solvent for compounds used in screen. Dissolves MTT precipitates to facilitate absorbance measurements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FGF-basic</td>
			<td style="width:237px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>Neural stem cell medium component.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelTrex</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A1413202</td>
			<td>Neural stem cell basement membrane matrix.&#160; Allows cells to attach to cell culture plates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gen5 3.04</td>
			<td style="width:237px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>Analysis software to determine cell counts for tdTomato expressing cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">25030081</td>
			<td>Neural stem cell medium component.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtest U-Bottom</td>
			<td style="width:237px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">3077</td>
			<td>Storage of compound libraries.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td style="width:119px;">M6494</td>
			<td>Active assay reagent to determine cellular viability.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multichannel pippette</td>
			<td style="width:237px;">Rainin</td>
			<td style="width:119px;">E8-1200</td>
			<td>Column-by-column addition of cell culture medium, MTT, or DMSO.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">21103049</td>
			<td>Neural stem cell base medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RFP filter cube</td>
			<td style="width:237px;">BioTek</td>
			<td align="right" style="width:119px;">1225103</td>
			<td>Filter in Cytation 5 used to image tdTomato expressing cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TrypLE</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12605036</td>
			<td>Cell dissociation reagent.</td>
		</tr>
	</tbody>
</table>

a strategy to identify compounds that affect cell growth and survival in cultured mammalian cells at low-to-moderate throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

The automated cell count data identified eleven compounds with less than 25% viability when normalized to the DMSO control while the MTT data identified these same compounds plus two additional ones (Table 1 and Table 2, shaded red). The two compounds found to be toxic only in the MTT assay (wells F3 and G10) had 31% and 39%, respectively, the number of tdTomato-positive cells as the control and by rank order were the next two most toxic compounds in this library after those deemed to be...

Watch this Scientific Journal Video about A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput at JoVE.com

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

This protocol offers a quick and efficient strategy to simultaneously use two assays to screen several hundred compounds for their effects on cell growth. The assays can provide us information on whether specific compounds are cytotoxic, inhibit cell growth, or enhance cell growth. By utilizing the two assays together, we are able to increase accuracy and identify cytotoxic compounds.In cases where the results of the two assays diverge, this strategy may allow us to identify compound with unique effects on cellular function that can be further examined by other means. The main advantage of this strategy is that it is quick, inexpensive, and non-labor intensive. Once mastered, it allows researchers to easily perform a primary screen of up to 200 compounds in one week, and then perform an exhaustive dose response curve on 10 to 15 compounds in another week.Demonstrating the procedure will be myself and Rohini Manickam, a staff scientist and post-baccalaureate fellow from the laboratory. First, in a biological safety cabinet, culture cells in a T75 flask with NSC medium so that they will be at least 80%confluent when beginning the assay. And place the flask in a cell culture incubator set at 37 degrees Celsius and five percent carbon dioxide.Once the cells reach 80%confluence, remove the flask from the incubator and aspirate off NSC medium. Add three milliliters of cell dissociation reagent and incubate for five minutes in the incubator. After incubation, add seven milliliters of NSC medium in the flask, and pipette vigorously to ensure all cells become detached.Transfer the dissociated cell solution to a 15 milliliter tube, and centrifuge at 200 times G for five minutes. Remove the supernatant from the tube and re-suspend the cells in 10 milliliters of NSC medium. Use an automated cell counter or hemocytometer to count the cells, and add NSC medium into the tube to readjust the concentration of cells to 200, 000 cells per milliliter.Then, using six of the eight slots of an eight channel multichannel pipettor to plate 100 microliters of the cell mixture column by column in 60 interior wells of three 96 well plates that have been coated by extracellular matrix. Add 100 microliters of base medium or NSC medium to all wells without cells to minimize potential evaporation from outermost wells containing cells. Under a cell culture microscope, visually inspect at least 10 wells on each of the three 96 well plates to confirm that the cells are seated at the expected density.Incubate at 37 degrees Celsius and five percent carbon dioxide. Remove the cell culture plates from the incubator 16 to 24 hours after splitting, and aspirate off NSC medium column by column with an eight channel multi-well pipettor, using only six of the eight multi-well slots. Add 95 microliters of fresh NSC medium to each well of the three replicate plates, and place the plates back into the incubator.With an eight channel multi-well pipettor, add 49 microliters of NSC medium into each of the interior 60 wells of an empty U-bottomed, V-bottomed, or round-bottomed 96 well plate. Unseal the prepared master compound plate. Pipette one microliter of the compound from the master plate into each of the interior 60 wells containing NSC medium, and mix the diluted compound three times with a bench top pipettor.Next, remove the three 96 well plates of NSCs from the incubator and dispense five microliters of the diluted compound into each well of the three plates that contain cells to yield a one to 1000 dilution for the final compound concentration of 10 micromolar with a DMSO concentration of 0.1%Incubate cells with compound for 72 hours, and proceed with cytotoxicity assays. To perform dose response assay, set up a 96 well. Use Column Two for six DMSO control replicates, and test triplicates of up to three different compounds at twofold serial dilutions, at six doses, starting with a high dose of 10 micromolar.Dilute four microliters of 100%DMSO or test compound in DMSO into 196 microliters of NSC medium in a 0.5 or 1.5 milliliter microcentrifuge tube. Add 25 microliters of the diluted DMSO to control wells, and 50 microliters of the diluted test compounds to their corresponding wells. Pipette 25 microliters of NSC medium to the remaining empty wells in the interior portion of the 96 well plate.With a multichannel pipettor, transfer 25 microliters of the compound from the 10 micromolar wells to the five micromolar wells, and mix at least five times. Replace the pipette tips, and repeat the process by mixing the remaining rows to generate triplicates at twofold dilutions for a total of six doses for each of the compounds. Transfer five microliters of each dilution into a new plate with culture cells that are being tested.Place the plate in the incubator at 37 degrees Celsius and five percent carbon dioxide. After cells have been incubated for 72 hours, remove the plate from the incubator and place it inside the plate reader. Open the imager software to set up protocol and experiment files for the study.Go to Imager Manual Mode on Task Manager and click Capture Now. Choose 96 well plate as the vessel type. Select 10 times for the magnification, and red fluorescent protein 531 and 593 for imaging tdTomato.Click on a well. Then click on Autofocus to focus image, and Auto Expose for proper exposure time. Manually adjust the focus and exposure if needed.Click the camera icon to capture the picture. Then click Process Analyze above image to continue building the protocol. And select the Analysis tab.To the right of the image, click Cellular Analysis in Add Analysis Step, and click Start. The image shows highlighted cells to indicate each individual cell. Click on the Options selection to alter parameters to better select cells based upon the fluorescence threshold or cell size.If the imager is properly counting the cells, then click Add Step at the bottom of the screen. Click the icon at the top of the screen to create experiment from the image set. And on the opened window, click Procedure under the Protocol tab.Then, in the new window that opens, select Read. Click Full Plate to select only the 60 wells that contain the cells. Click OK to save changes.Then click OK in the Procedure window. Click the Play icon to run the plate. In the prompt, save the file, and then in the Load Plate prompt, click OK.Upon completion of the imaging, download the cell count data from the imager software program to a spreadsheet for analysis.Within two hours of completing tdTomato imaging, begin the MTT assay. Weigh out 25 milligrams of MTT and re-suspend it in five milliliters of NSC medium to make five milligrams per milliliter MTT stock solution. Vortex the solution for several minutes, until there are no visible precipitates of MTT.Remove cell culture plates from the incubator and aspirate off cell culture medium. Dilute MTT one to 10 in fresh NSC medium, and add 100 microliters of the diluted MTT to each well of cells. Incubate cells at 37 degrees Celsius for two hours.A purplish precipitate is visible roughly in proportion to the number of cells in the well. Aspirate the MTT solution off the plates, and add 50 microliters of 100%DMSO to each well. Place the plates on a shaker at room temperature for 10 minutes at 400 RPM.After that, transfer the plate to a plate reader. Read the absorbance of each well at the preset wavelength of 595 nanometers, and export data to spreadsheet for analysis, exactly as for the cell count data. Examination of tdTomato fluorescence images revealed two wells discordant for toxicity between the MTT and cell count assay.For one well, cell count data suggested no toxicity. But MTT data did. Another well had overestimated cell count by MTT relative to tdTomato count, and appeared to have larger cells, whereas in wells where MTT underestimated cell count relative to tdTomato, the cells appeared to be smaller.In summary, the tdTomato assay classified 11 compounds as toxic, 11 as growth inhibitory, and one as growth enhancing, with 34 having no apparent effect on cell growth. The MTT assay classified 13 compounds as toxic, two as growth inhibitory, and one as growth enhancing, with 41 having no apparent effect on cell growth. The graph of viable cells for the compound WP1066 shows a relatively flat curve with no toxicity for the four lowest concentrations.The curve falls rapidly at the five micromolar dose, and drops to nearly full toxicity at 10 micromolar, which leads to the lethal dose 50 as 4.4 and 6.0 micromolar for the tdTomato assay and the MTT assay. It is critical that compounds are properly diluted in the dose response screen so that the correct dose response curve is generated. Compounds identified by this procedure to affect cell growth can be further characterized by functional assays to identify potentially novel signaling pathways that control cell growth.Compounds that are toxic to neural stem cells have been tested as potential therapeutic agents for brain cancers, such as glioblastoma and neuroblastoma. Always wear gloves and a lab coat when handling mammalian cells, DMSO, and MTT. Dispose DMSO and MTT according to the Chemical Waste Guidelines of your institution.

a-strategy-to-identify-compounds-that-affect-cell-growth-survival

Research

JoVE Journal

Biochemistry

8.4K visualizações.  National Institutes of Health (NIH). Este protocolo oferece uma estratégia rápida e eficiente para usar simultaneamente dois ensaios para triagem de várias centenas de compostos para seus efeitos no crescimento celular. Os ensaios podem nos fornecer informações sobre se compostos específicos são citotóxicos, inibem o crescimento celular ou melhoram o crescimento celular. Utilizando os dois ensaios juntos, somos capazes de aumentar a precisão e identificar compostos citotóxicos.Nos casos em que os resultados dos ...